NOTCH1 inhibition induces apoptosis of mammary tumor cells. (A) NOTCH1 expression is rapidly suppressed in doxycycline-treated mammary tumor cell lines. Cell lines were left untreated or treated with doxycycline (2 μg/ml) for 24 hours, and lysates were analyzed for ICN1 expression with immunoblotting. A doxycycline-regulated T-ALL cell line 3404 is included as a control. (B) Mammary tumor cell lines express cytokeratin 8/18, but have variable expression of basal cytokeratins 5 and 14. Cell-line lysates were analyzed for CK8/18, CK5, and CK14 expression with immunoblotting. (C) NOTCH1 inhibition arrests mammary tumor cell growth. Three mammary tumor cell lines (8066, 8534, and 8542) and T-ALL cell line 3404 were left untreated or treated with doxycycline (2 μg/ml) for 72 hours, and cell growth was measured by using an MTT assay. The figure represents the average of three independent experiments, statistically analyzed by the Student paired t test. (D) NOTCH1 inhibition induces apoptosis of multiple mammary tumor cell lines. Tumor-derived cell lines 8066, 8534, and 8542 were treated with 2 μg/ml doxycycline or left untreated for 0, 24, or 72 hours. Cells were assayed for DNA content by staining with PI followed by flow cytometry. (E) NOTCH1 inhibition results in caspase 3 activation. Cells were left untreated or treated with doxycycline (2 μg/ml) for 72 hours, and lysates were analyzed for cleaved or total caspase-3 levels with immunoblotting.